Abstract
Primary human hepatocytes (PHHs) are used extensively for in vitro liver cultures to study hepatic functions. However, limited availability and invasive retrieval prevent their widespread use. Induced pluripotent stem cells exhibit significant potential since they can be obtained non-invasively and differentiated into hepatic lineages, such as hepatocyte-like cells (iHLCs). However, there are concerns about their fetal phenotypic characteristics and their hepatic functions compared to PHHs in culture. Therefore, we performed an RNA-sequencing (RNA-seq) analysis to understand pathways that are either up- or downregulated in each cell type. Analysis of the RNA-seq data showed an upregulation in the bile secretion pathway where genes such as AQP9 and UGT1A1 were higher expressed in PHHs compared to iHLCs by 455- and 15-fold, respectively. Upon immunostaining, bile canaliculi were shown to be present in PHHs. The TCA cycle in PHHs was upregulated compared to iHLCs. Cellular analysis showed a 2–2.5-fold increase in normalized urea production in PHHs compared to iHLCs. In addition, drug metabolism pathways, including cytochrome P450 (CYP450) and UDP-glucuronosyltransferase enzymes, were upregulated in PHHs compared to iHLCs. Of note, CYP2E1 gene expression was significantly higher (21,810-fold) in PHHs. Acetaminophen and ethanol were administered to PHH and iHLC cultures to investigate differences in biotransformation. CYP450 activity of baseline and toxicant-treated samples was significantly higher in PHHs compared to iHLCs. Our analysis revealed that iHLCs have substantial differences from PHHs in critical hepatic functions. These results have highlighted the differences in gene expression and hepatic functions between PHHs and iHLCs to motivate future investigation.
Graphical Abstract
Characterizing the transcriptomic differences between primary human hepatocytes (PHHs) and induced pluripotent stem cell hepatocyte-like cells (iHLCs) using RNA-sequencing and phenotypic measurements
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Availability of data and materials
We will make the RNA-seq data available in the Gene Expression Omnibus upon publication. All experimental supplies were obtained from commercial vendors.
Abbreviations
- HFC:
-
7-Hydroxy-4-trifluoromethylcoumarin
- MFC:
-
7-Methoxy-4-trifluoromethylcoumarin
- APAP:
-
Acetaminophen
- AQP9:
-
Aquaporin 9
- ABC:
-
ATP binding cassette
- BMP-4:
-
Bone morphogenic factor-4
- BSA:
-
Bovine serum albumin
- CS:
-
Collagen sandwich
- CYP450:
-
Cytochrome P450
- ESCs:
-
Embryonic stem cells
- ER:
-
Endoplasmic reticulum
- EtOH:
-
Ethanol
- EDTA:
-
Ethylenediaminetetraacetic acid
- FXR:
-
Farnesoid X receptor
- FGF-2:
-
Fibroblast growth factor-2
- GSH:
-
Glutathione
- HGF:
-
Hepatocyte growth factor
- iHLCs:
-
Hepatocyte-like cells
- iPSCs:
-
Induced pluripotent stem cells
- LC50 :
-
Lethal concentration 50
- LSECs:
-
Liver sinusoidal endothelial cells
- mTOR:
-
Mammalian target of rapamycin
- NPCs:
-
Non-parenchymal cells
- OGDHL:
-
Oxoglutarate dehydrogenase L
- PBS:
-
Phosphate buffered saline
- PCA:
-
Principal component analyses
- PCK1:
-
Phosphoenolpyruvate carboxykinase 1
- PI3K:
-
Phosphoinositide 3-kinase
- PHHs:
-
Primary human hepatocytes
- AKT:
-
Protein kinase B
- ROS:
-
Reactive oxygen species
- SDS:
-
Sodium dodecyl sulfate
- TCPS:
-
Tissue culture polystyrene
- t-SNE:
-
T-distributed stochastic neighbor embedding
- UGT:
-
UDP-glucuronosyltransferase
References
Akram M (2014) Citric acid cycle and role of its intermediates in metabolism. Cell Biochem Biophys 68(3):475–478. https://doi.org/10.1007/s12013-013-9750-1
Allain EP, Rouleau M, Lévesque E, Guillemette C (2020) Emerging roles for UDP-glucuronosyltransferases in drug resistance and cancer progression. Br J Cancer 122(9):1277–1287. https://doi.org/10.1038/s41416-019-0722-0
Anand U, Anand CV (1999) Connecting links between the urea cycle and the TCA cycle: a tutorial exercise. Biochemical Education 27(3):153–154. https://doi.org/10.1016/S0307-4412(99)00041-2
Ardalani H, Sengupta S, Harms V, Vickerman V, Thomson JA, Murphy WL (2019) 3-D culture and endothelial cells improve maturity of human pluripotent stem cell-derived hepatocytes. Acta Biomater 95:371–381. https://doi.org/10.1016/j.actbio.2019.07.047
Arias IM, Alter HJ, Boyer JL, Cohen DE, Shafritz DA, Thorgeirsson SS, Wolkoff AW (2009) The Liver: Biology and Pathobiology, 5th edn. Wiley, Hoboken, NJ. ISBN: 9780470723135
Barbier O, Trottier J, Kaeding J, Caron P, Verreault M (2009) Lipid-activated transcription factors control bile acid glucuronidation. Mol Cell Biochem 326(1–2):3–8. https://doi.org/10.1007/s11010-008-0001-5
Brazovskaja A, Treutlein B, Camp JG (2019) High-throughput single-cell transcriptomics on organoids. Curr Opin Biotechnol 55:167–171. https://doi.org/10.1016/j.copbio.2018.11.002
Camp JG, Sekine K, Gerber T, Loeffler-Wirth H, Binder H, Gac M, Kanton S, Kageyama J, Damm G, Seehofer D, Belicova L, Bickle M, Barsacchi R, Okuda R, Yoshizawa E, Kimura M, Ayabe H, Taniguchi H, Takebe T, Treutlein B (2017) Multilineage communication regulates human liver bud development from pluripotency. Nature 546(7659):533–538. https://doi.org/10.1038/nature22796
Chen T, Oh S, Gregory S, Shen X, Diehl AM (2020) Single-cell omics analysis reveals functional diversification of hepatocytes during liver regeneration. JCI Insight 5(22). https://doi.org/10.1172/jci.insight.141024
Chiang JYL (2013) Bile acid metabolism and signaling. Compr Physiol 3(3):1191–1212. https://doi.org/10.1002/cphy.c120023
Choudhury Y, Toh YC, Xing J, Qu Y, Poh J, Li H, Tan HS, Kanesvaran R, Yu H, Tan M-H (2017) Patient-specific hepatocyte-like cells derived from induced pluripotent stem cells model pazopanib-mediated hepatotoxicity. Sci Rep 7(1):41238. https://doi.org/10.1038/srep41238
Dao Thi VL, Wu X, Belote RL, Andreo U, Takacs CN, Fernandez JP, Vale-Silva LA, Prallet S, Decker CC, Fu RM, Qu B, Uryu K, Molina H, Saeed M, Steinmann E, Urban S, Singaraja RR, Schneider WM, Simon SM, Rice CM (2020) Stem cell-derived polarized hepatocytes. Nat Commun 11(1):1677. https://doi.org/10.1038/s41467-020-15337-2
David S, Hamilton JP (2010) Drug-induced liver injury. US Gastroenterol Hepatol Rev 6:73–80
de Cima S, Polo LM, Díez-Fernández C, Martínez AI, Cervera J, Fita I, Rubio V (2015) Structure of human carbamoyl phosphate synthetase: deciphering the on/off switch of human ureagenesis. Sci Rep 5(1):16950. https://doi.org/10.1038/srep16950
Detzel CJ, Kim Y, Rajagopalan P (2011) Engineered three-dimensional liver mimics recapitulate critical rat-specific bile acid pathways. Tissue Eng Part A 17(5–6):677–689. https://doi.org/10.1089/ten.TEA.2010.0423
Donato MT, Jiménez N, Castell JV, Gómez-Lechón MJ (2004) Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes. Drug Metab Dispos 32(7):699–706. https://doi.org/10.1124/dmd.32.7.699
Du Y, Wang J, Jia J, Song N, Xiang C, Xu J, Hou Z, Su X, Liu B, Jiang T, Zhao D, Sun Y, Shu J, Guo Q, Yin M, Sun D, Lu S, Shi Y, Deng H (2014) Human hepatocytes with drug metabolic function induced from fibroblasts by lineage reprogramming. Cell Stem Cell 14(3):394–403. https://doi.org/10.1016/j.stem.2014.01.008
Du C, Feng Y, Qiu D, Xu Y, Pang M, Cai N, Xiang AP, Zhang Q (2018) Highly efficient and expedited hepatic differentiation from human pluripotent stem cells by pure small-molecule cocktails. Stem Cell Res Ther 9(1):58. https://doi.org/10.1186/s13287-018-0794-4
Dunn JC, Yarmush ML, Koebe HG, Tompkins RG (1989) Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration. FASEB J 3(2):174–177
Gao X, Liu Y (2017) A transcriptomic study suggesting human iPSC-derived hepatocytes potentially offer a better in vitro model of hepatotoxicity than most hepatoma cell lines. Cell Biol Toxicol 33(4):407–421. https://doi.org/10.1007/s10565-017-9383-z
Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, Bode JG, Bolleyn J, Borner C, Böttger J, Braeuning A, Budinsky RA, Burkhardt B, Cameron NR, Camussi G, Cho C-S, Choi Y-J, Craig Rowlands J, Dahmen U, ... Hengstler JG (2013) Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol 87(8):1315–1530. https://doi.org/10.1007/s00204-013-1078-5
Grant R, Hallett J, Forbes S, Hay D, Callanan A (2019) Blended electrospinning with human liver extracellular matrix for engineering new hepatic microenvironments. Sci Rep 9(1):6293. https://doi.org/10.1038/s41598-019-42627-7
Guo C, Sun L, Chen X, Zhang D (2013) Oxidative stress, mitochondrial damage and neurodegenerative diseases. Neural Regen Res 8(21):2003–2014. https://doi.org/10.3969/j.issn.1673-5374.2013.21.009
Gupta R, Schrooders Y, Hauser D, van Herwijnen M, Albrecht W, Ter Braak B, Brecklinghaus T, Castell JV, Elenschneider L, Escher S, Guye P, Hengstler JG, Ghallab A, Hansen T, Leist M, Maclennan R, Moritz W, Tolosa L, Tricot T, ... Caiment F (2021) Comparing in vitro human liver models to in vivo human liver using RNA-Seq. Arch Toxicol 95(2):573–589. https://doi.org/10.1007/s00204-020-02937-6
Guttman Y, Nudel A, Kerem Z (2019) Polymorphism in cytochrome P450 3A4 is ethnicity related. Front Genet 10:224. https://doi.org/10.3389/fgene.2019.00224
Harris MA, Clark J, Ireland A, Lomax J, Ashburner M, Foulger R, Eilbeck K, Lewis S, Marshall B, Mungall C, Richter J, Rubin GM, Blake JA, Bult C, Dolan M, Drabkin H, Eppig JT, Hill DP, Ni L, ..., White R (2004) The Gene Ontology (GO) database and informatics resource. Nucleic Acids Res 32(Database issue):D258-261. https://doi.org/10.1093/nar/gkh036
Herrero J, Muffato M, Beal K, Fitzgerald S, Gordon L, Pignatelli M, Vilella AJ, Searle SM, Amode R, Brent S, Spooner W, Kulesha E, Yates A, Flicek P (2016) Ensembl comparative genomics resources. Database (Oxford) 2016:bav096. https://doi.org/10.1093/database/bav096. Erratum in: Database (Oxford). 2016;2016. pii: baw053. https://doi.org/10.1093/database/baw053. PMID: 26896847; PMCID: PMC4761110
Jansen PL (2017) Fibroblast growth factor 19, a double-edged sword. Hepat Oncol 4(1):1–4. https://doi.org/10.2217/hep-2017-0008
Jelen S, Gena P, Lebeck J, Rojek A, Praetorius J, Frøkiaer J, Fenton RA, Nielsen S, Calamita G, Rützler M (2012) Aquaporin-9 and urea transporter-A gene deletions affect urea transmembrane passage in murine hepatocytes. Am J Physiol Gastrointest Liver Physiol 303(11):G1279-1287. https://doi.org/10.1152/ajpgi.00153.2012
Jolliffe IT (2002) Principal component analysis for special types of data. In: Jolliffe IT (ed) Principal component analysis. Springer, New York, pp 338–372. https://doi.org/10.1007/0-387-22440-8_13
Kaserman JE, Wilson AA (2017) Protocol for directed differentiation of human induced pluripotent stem cells (iPSCs) to a hepatic lineage. Methods Mol Biol 1639:151–160. https://doi.org/10.1007/978-1-4939-7163-3_15
Kim Y, Rajagopalan P (2010) 3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes. PLoS ONE 5(11):e15456. https://doi.org/10.1371/journal.pone.0015456
Kim Y, Larkin AL, Davis RM, Rajagopalan P (2010) The design of in vitro liver sinusoid mimics using chitosan-hyaluronic acid polyelectrolyte multilayers. Tissue Eng Part A 16(9):2731–2741. https://doi.org/10.1089/ten.tea.2009.0695
Kim D, Langmead B, Salzberg SL (2015) HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12(4):357–360. https://doi.org/10.1038/nmeth.3317
Klaassen CD (2013) Casarett and Doull’s toxicology: the basic science of poisons (Vol. 1236), vol 1236. McGraw-Hill, New York
Klein K, Winter S, Turpeinen M, Schwab M, Zanger UM (2010) Pathway-targeted pharmacogenomics of CYP1A2 in human liver. Front Pharmacol 1:129–129. https://doi.org/10.3389/fphar.2010.00129
Kocarek TA, Zangar RC, Novak RF (2000) Post-transcriptional regulation of rat CYP2E1 expression: role of CYP2E1 mRNA untranslated regions in control of translational efficiency and message stability. Arch Biochem Biophys 376(1):180–190. https://doi.org/10.1006/abbi.2000.1704
Kroll T, Prescher M, Smits SHJ, Schmitt L (2021) Structure and function of hepatobiliary ATP binding cassette transporters. Chem Rev 121(9):5240–5288. https://doi.org/10.1021/acs.chemrev.0c00659
Krumm J, Sekine K, Samaras P, Brazovskaja A, Breunig M, Yasui R, Kleger A, Taniguchi H, Wilhelm M, Treutlein B, Camp JG, Kuster B (2022) High temporal resolution proteome and phosphoproteome profiling of stem cell-derived hepatocyte development. Cell Rep 38(13):110604. https://doi.org/10.1016/j.celrep.2022.110604
Kuna L, Bozic I, Kizivat T, Bojanic K, Mrso M, Kralj E, Smolic R, Wu GY, Smolic M (2018) Models of drug induced liver injury (DILI) - current issues and future perspectives. Curr Drug Metab 19(10):830–838. https://doi.org/10.2174/1389200219666180523095355
Larkin AL, Rodrigues RR, Murali TM, Rajagopalan P (2013) Designing a multicellular organotypic 3D liver model with a detachable, nanoscale polymeric Space of Disse. Tissue Eng Part C Methods 19(11):875–884. https://doi.org/10.1089/ten.TEC.2012.0700
Laudadio I, Manfroid I, Achouri Y, Schmidt D, Wilson MD, Cordi S, Thorrez L, Knoops L, Jacquemin P, Schuit F, Pierreux CE, Odom DT, Peers B, Lemaigre FP (2012) A feedback loop between the liver-enriched transcription factor network and miR-122 controls hepatocyte differentiation. Gastroenterology 142(1):119–129. https://doi.org/10.1053/j.gastro.2011.09.001
Laurens van der Maaten GH (2008) Visualizing data using t-SNE. J Mach Learn Res 9(86):2579–2605
Lauschke VM, Hendriks DF, Bell CC, Andersson TB, Ingelman-Sundberg M (2016) Novel 3D culture systems for studies of human liver function and assessments of the hepatotoxicity of drugs and drug candidates. Chem Res Toxicol 29(12):1936–1955. https://doi.org/10.1021/acs.chemrestox.6b00150
Leung TM, Nieto N (2013) CYP2E1 and oxidant stress in alcoholic and non-alcoholic fatty liver disease. J Hepatol 58(2):395–398. https://doi.org/10.1016/j.jhep.2012.08.018
Liao Y, Smyth GK, Shi W (2014) featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30(7):923–930. https://doi.org/10.1093/bioinformatics/btt656
Lindley C, Hamilton G, McCune JS, Faucette S, Shord SS, Hawke RL, Wang H, Gilbert D, Jolley S, Yan B, LeCluyse EL (2002) The effect of cyclophosphamide with and without dexamethasone on cytochrome P450 3A4 and 2B6 in human hepatocytes. Drug Metab Dispos 30(7):814–822. https://doi.org/10.1124/dmd.30.7.814
Liu L-G, Yan H, Yao P, Zhang W, Zou L-J, Song F-F, Li K, Sun X-F (2005) CYP2E1-dependent hepatotoxicity and oxidative damage after ethanol administration in human primary hepatocytes. World J Gastroenterol 11(29):4530–4535. https://doi.org/10.3748/wjg.v11.i29.4530
Liu D, Yu Q, Li Z, Zhang L, Hu M, Wang C, Liu Z (2021) UGT1A1 dysfunction increases liver burden and aggravates hepatocyte damage caused by long-term bilirubin metabolism disorder. Biochem Pharmacol 190:114592. https://doi.org/10.1016/j.bcp.2021.114592
Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15(12):550. https://doi.org/10.1186/s13059-014-0550-8
Lu Y, Cederbaum AI (2008) CYP2E1 and oxidative liver injury by alcohol. Free Radical Biol Med 44(5):723–738. https://doi.org/10.1016/j.freeradbiomed.2007.11.004
Madhunapantula SV, Mosca PJ, Robertson GP (2011) The Akt signaling pathway: an emerging therapeutic target in malignant melanoma. Cancer Biol Ther 12(12):1032–1049. https://doi.org/10.4161/cbt.12.12.18442
Mallanna SK, Duncan SA (2013) Differentiation of hepatocytes from pluripotent stem cells. Curr Protoc Stem Cell Biol 26:1g.4.1-1g.4.13. https://doi.org/10.1002/9780470151808.sc01g04s26
Manley S, Ding W (2015) Role of farnesoid X receptor and bile acids in alcoholic liver disease. Acta Pharm Sin B 5(2):158–167. https://doi.org/10.1016/j.apsb.2014.12.011
Marinelli RA, Lehmann GL, Soria LR, Marchissio MJ (2011) Hepatocyte aquaporins in bile formation and cholestasis. Front Biosci (landmark Ed) 16(7):2642–2652. https://doi.org/10.2741/3877
Maruo Y, Iwai M, Mori A, Sato H, Takeuchi Y (2005) Polymorphism of UDP-glucuronosyltransferase and drug metabolism. Curr Drug Metab 6(2):91–99. https://doi.org/10.2174/1389200053586064
Montal ED, Bhalla K, Dewi RE, Ruiz CF, Haley JA, Ropell AE, Gordon C, Haley JD, Girnun GD (2019) Inhibition of phosphoenolpyruvate carboxykinase blocks lactate utilization and impairs tumor growth in colorectal cancer. Cancer Metab 7(1):8. https://doi.org/10.1186/s40170-019-0199-6
Mora C, Serzanti M, Consiglio A, Memo M, Dell’Era P (2017) Clinical potentials of human pluripotent stem cells. Cell Biol Toxicol 33(4):351–360. https://doi.org/10.1007/s10565-017-9384-y
Nakamori D, Akamine H, Takayama K, Sakurai F, Mizuguchi H (2017) Direct conversion of human fibroblasts into hepatocyte-like cells by ATF5, PROX1, FOXA2, FOXA3, and HNF4A transduction. Sci Rep 7(1):16675. https://doi.org/10.1038/s41598-017-16856-7
Nell P, Kattler K, Feuerborn D, Hellwig B, Rieck A, Salhab A, Lepikhov K, Gasparoni G, Thomitzek A, Belgasmi K, Blüthgen N, Morkel M, Küppers-Munther B, Godoy P, Hay DC, Cadenas C, Marchan R, Vartak N, Edlund K, Hengstler JG (2022) Identification of an FXR-modulated liver-intestine hybrid state in iPSC-derived hepatocyte-like cells. J Hepatol 77(5):1386–1398. https://doi.org/10.1016/j.jhep.2022.07.009
Neve EP, Ingelman-Sundberg M (2000) Molecular basis for the transport of cytochrome P450 2E1 to the plasma membrane. J Biol Chem 275(22):17130–17135. https://doi.org/10.1074/jbc.M000957200
Novak RF, Woodcroft KJ (2000) The alcohol-inducible form of cytochrome P450 (CYP 2E1): role in toxicology and regulation of expression. Arch Pharm Res 23(4):267–282. https://doi.org/10.1007/bf02975435
Orbach SM, Cassin ME, Ehrich MF, Rajagopalan P (2017) Investigating acetaminophen hepatotoxicity in multi-cellular organotypic liver models. In Vitro Toxicol 42:10–20. https://doi.org/10.1016/j.tiv.2017.03.008
Orbach SM, Ehrich MF, Rajagopalan P (2018) High-throughput toxicity testing of chemicals and mixtures in organotypic multi-cellular cultures of primary human hepatic cells. In Vitro Toxicol 51:83–94. https://doi.org/10.1016/j.tiv.2018.05.006
Orge ID, Gadd VL, Barouh JL, Rossi EA, Carvalho RH, Smith I, Allahdadi KJ, Paredes BD, Silva DN, Damasceno PKF, Sampaio GL, Forbes SJ, Soares MBP, Souza BS, d. F. (2020) Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells. Stem Cell Res Ther 11(1):154. https://doi.org/10.1186/s13287-020-01665-z
Overeem AW, Klappe K, Parisi S, Klöters-Planchy P, Mataković L, du Teil Espina M, Drouin CA, Weiss KH, van, I. S. C. D. (2019) Pluripotent stem cell-derived bile canaliculi-forming hepatocytes to study genetic liver diseases involving hepatocyte polarity. J Hepatol 71(2):344–356. https://doi.org/10.1016/j.jhep.2019.03.031
Perugorria MJ, Olaizola P, Labiano I, Esparza-Baquer A, Marzioni M, Marin JJG, Bujanda L, Banales JM (2019) Wnt–β-catenin signalling in liver development, health and disease. Nat Rev Gastroenterol Hepatol 16(2):121–136. https://doi.org/10.1038/s41575-018-0075-9
Pettinato G, Lehoux S, Ramanathan R, Salem MM, He L-X, Muse O, Flaumenhaft R, Thompson MT, Rouse EA, Cummings RD, Wen X, Fisher RA (2019) Generation of fully functional hepatocyte-like organoids from human induced pluripotent stem cells mixed with endothelial cells. Sci Rep 9(1):8920. https://doi.org/10.1038/s41598-019-45514-3
Preissner SC, Hoffmann MF, Preissner R, Dunkel M, Gewiess A, Preissner S (2013) Polymorphic cytochrome P450 enzymes (CYPs) and their role in personalized therapy. PLoS ONE 8(12):e82562. https://doi.org/10.1371/journal.pone.0082562
Ramli MNB, Lim YS, Koe CT, Demircioglu D, Tng W, Gonzales KAU, Tan CP, Szczerbinska I, Liang H, Soe EL, Lu Z, Ariyachet C, Yu KM, Koh SH, Yaw LP, Jumat NHB, Lim JSY, Wright G, Shabbir A, ... Chan Y-S (2020) Human pluripotent stem cell-derived organoids as models of liver disease. Gastroenterology 159(4):1471–1486. https://doi.org/10.1053/j.gastro.2020.06.010
Rowe RG, Daley GQ (2019) Induced pluripotent stem cells in disease modelling and drug discovery. Nat Rev Genet 20(7):377–388. https://doi.org/10.1038/s41576-019-0100-z
Rowland A, Miners JO, Mackenzie PI (2013) The UDP-glucuronosyltransferases: their role in drug metabolism and detoxification. Int J Biochem Cell Biology 45(6):1121–1132. https://doi.org/10.1016/j.biocel.2013.02.019
Roy-Chowdhury N, Wang X, Guha C, Roy-Chowdhury J (2017) Hepatocyte-like cells derived from induced pluripotent stem cells. Hepatol Int 11(1):54–69. https://doi.org/10.1007/s12072-016-9757-y
Rui L (2014) Energy metabolism in the liver. Compr Physiol 4(1):177–197. https://doi.org/10.1002/cphy.c130024
Sauer V, Roy-Chowdhury N, Guha C, Roy-Chowdhury J (2014) Induced pluripotent stem cells as a source of hepatocytes. Curr Pathobiol Rep 2(1):11–20. https://doi.org/10.1007/s40139-013-0039-2
Shambaugh GE 3rd (1977) Urea biosynthesis I. The urea cycle and relationships to the citric acid cycle. Am J Clin Nutr 30(12):2083–2087. https://doi.org/10.1093/ajcn/30.12.2083
Shi Y, Inoue H, Wu JC, Yamanaka S (2017) Induced pluripotent stem cell technology: a decade of progress. Nat Rev Drug Discovery 16(2):115–130. https://doi.org/10.1038/nrd.2016.245
Shinozawa T, Kimura M, Cai Y, Saiki N, Yoneyama Y, Ouchi R, Koike H, Maezawa M, Zhang RR, Dunn A, Ferguson A, Togo S, Lewis K, Thompson WL, Asai A, Takebe T (2021) High-fidelity drug-induced liver injury screen using human pluripotent stem cell-derived organoids. Gastroenterology 160(3):831-846.e810. https://doi.org/10.1053/j.gastro.2020.10.002
Si-Tayeb K, Noto FK, Nagaoka M, Li J, Battle MA, Duris C, North PE, Dalton S, Duncan SA (2010) Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 51(1):297–305. https://doi.org/10.1002/hep.23354
Sivandzade F, Bhalerao A, Cucullo L (2019) Analysis of the mitochondrial membrane potential using the cationic JC-1 dye as a sensitive fluorescent probe. Bio Protoc 9(1). https://doi.org/10.21769/BioProtoc.3128
Sjogren A-KM, Liljevald M, Glinghammar B, Sagemark J, Li X-Q, Jonebring A, Cotgreave I, Brolén G, Andersson TB (2014) Critical differences in toxicity mechanisms in induced pluripotent stem cell-derived hepatocytes, hepatic cell lines and primary hepatocytes. Arch Toxicol 88(7):1427–1437. https://doi.org/10.1007/s00204-014-1265-z
Song Z, Cai J, Liu Y, Zhao D, Yong J, Duo S, Song X, Guo Y, Zhao Y, Qin H, Yin X, Wu C, Che J, Lu S, Ding M, Deng H (2009) Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells. Cell Res 19(11):1233–1242. https://doi.org/10.1038/cr.2009.107
Stark R, Pasquel F, Turcu A, Pongratz RL, Roden M, Cline GW, Shulman GI, Kibbey RG (2009) Phosphoenolpyruvate cycling via mitochondrial phosphoenolpyruvate carboxykinase links anaplerosis and mitochondrial GTP with insulin secretion. J Biol Chem 284(39):26578–26590. https://doi.org/10.1074/jbc.M109.011775
Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4):663–676. https://doi.org/10.1016/j.cell.2006.07.024
Takebe T, Sekine K, Enomura M, Koike H, Kimura M, Ogaeri T, Zhang R-R, Ueno Y, Zheng Y-W, Koike N, Aoyama S, Adachi Y, Taniguchi H (2013) Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 499(7459):481–484. https://doi.org/10.1038/nature12271
Tegge AN, Rodrigues RR, Larkin AL, Vu L, Murali TM, Rajagopalan P (2018) Transcriptomic analysis of hepatic cells in multicellular organotypic liver models. Sci Rep 8(1):11306. https://doi.org/10.1038/s41598-018-29455-x
Tzanakakis ES, Hansen LK, Hu WS (2001) The role of actin filaments and microtubules in hepatocyte spheroid self-assembly. Cell Motil Cytoskeleton 48(3):175–189. https://doi.org/10.1002/1097-0169(200103)48:3%3c175::Aid-cm1007%3e3.0.Co;2-2
Viiri LE, Rantapero T, Kiamehr M, Alexanova A, Oittinen M, Viiri K, Niskanen H, Nykter M, Kaikkonen MU, Aalto-Setälä K (2019) Extensive reprogramming of the nascent transcriptome during iPSC to hepatocyte differentiation. Sci Rep 9(1):3562. https://doi.org/10.1038/s41598-019-39215-0
Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10(1):57–63. https://doi.org/10.1038/nrg2484
Wang J, Zhao P, Wan Z, Jin X, Cheng Y, Yan T, Qing S, Ding N, Xin S (2016) Differentiation of human foreskin fibroblast-derived induced pluripotent stem cells into hepatocyte-like cells. Cell Biochem Funct 34(7):475–482. https://doi.org/10.1002/cbf.3210
Wang G, Zheng Y, Wang Y, Cai Z, Liao N, Liu J, Zhang W (2018) Co-culture system of hepatocytes and endothelial cells: two in vitro approaches for enhancing liver-specific functions of hepatocytes. Cytotechnology 70(4):1279–1290. https://doi.org/10.1007/s10616-018-0219-3
Wang Z, Dong C (2019) Gluconeogenesis in cancer: function and regulation of PEPCK, FBPase, and G6Pase. Trends in Cancer 5(1):30–45. https://doi.org/10.1016/j.trecan.2018.11.003
Wesley BT, Ross ADB, Muraro D, Miao Z, Saxton S, Tomaz RA, Morell CM, Ridley K, Zacharis ED, Petrus-Reurer S, Kraiczy J, Mahbubani KT, Brown S, Garcia-Bernardo J, Alsinet C, Gaffney D, Horsfall D, Tysoe OC, Botting RA, ... Vallier L (2022) Single-cell atlas of human liver development reveals pathways directing hepatic cell fates. Nat Cell Biol 24(10):1487–1498. https://doi.org/10.1038/s41556-022-00989-7
Wickham H (2011) ggplot2. Wiley Interdisciplinary Reviews: Computational Statistics 3(2):180–185
Wills LR, Rajagopalan P (2020) Advances in human induced pluripotent stem cell-derived hepatocytes for use in toxicity testing. Ann Biomed Eng 48(3):1045–1057. https://doi.org/10.1007/s10439-019-02331-z
Xiang J, Wang K, Tang N (2023) PCK1 dysregulation in cancer: metabolic reprogramming, oncogenic activation, and therapeutic opportunities. Genes Dis 10(1):101–112. https://doi.org/10.1016/j.gendis.2022.02.010
Xie Y, Yao J, Jin W, Ren L, Li X (2021) Induction and maturation of hepatocyte-like cells in vitro: focus on technological advances and challenges [Review]. Front Cell Dev Biol 9. https://doi.org/10.3389/fcell.2021.765980
Xu Z, He X, Shi X, Xia Y, Liu X, Wu H, Li P, Zhang H, Yin W, Du X, Li L, Li Y (2018) Analysis of differentially expressed genes among human hair follicle–derived iPSCs, induced hepatocyte-like cells, and primary hepatocytes. Stem Cell Res Ther 9(1):211. https://doi.org/10.1186/s13287-018-0940-z
Yu G, Wang LG, Han Y, He QY (2012) clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16(5):284–287. https://doi.org/10.1089/omi.2011.0118
Yu S, Meng S, Xiang M, Ma H (2021) Phosphoenolpyruvate carboxykinase in cell metabolism: roles and mechanisms beyond gluconeogenesis. Mol Metab 53:101257. https://doi.org/10.1016/j.molmet.2021.101257
Zanger UM, Schwab M (2013) Cytochrome P450 enzymes in drug metabolism: regulation of gene expression, enzyme activities, and impact of genetic variation. Pharmacol Ther 138(1):103–141. https://doi.org/10.1016/j.pharmthera.2012.12.007
Zeilinger K, Freyer N, Damm G, Seehofer D, Knöspel F (2016) Cell sources for in vitro human liver cell culture models. Exp Biol Med (maywood) 241(15):1684–1698. https://doi.org/10.1177/1535370216657448
Zhang X, Jonassen I (2020) RASflow: an RNA-Seq analysis workflow with Snakemake. BMC Bioinformatics 21(1):110. https://doi.org/10.1186/s12859-020-3433-x
Zhang W, Li W, Liu B, Wang P, Li W, Zhang H (2012) Efficient generation of functional hepatocyte-like cells from human fetal hepatic progenitor cells in vitro. J Cell Physiol 227(5):2051–2058. https://doi.org/10.1002/jcp.22934
Zhou SF, Xue CC, Yu XQ, Li C, Wang G (2007) Clinically important drug interactions potentially involving mechanism-based inhibition of cytochrome P450 3A4 and the role of therapeutic drug monitoring. Ther Drug Monit 29(6):687–710. https://doi.org/10.1097/FTD.0b013e31815c16f5
Zhu X, Zhang B, He Y, Bao J (2021) Liver organoids: formation strategies and biomedical applications. Tissue Eng Regen Med 18(4):573–585. https://doi.org/10.1007/s13770-021-00357-w
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Figures 1, 4c/d, and 5a/f were created using Biorender.com.
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The authors gratefully acknowledge support for this work from the National Science Foundation (NSF DBI-1759858, T.M.M., K.A., and Y.S.; DBI-2233967, T.M.M. and Y.S.; NSF 2200045, T.M.M. and P.R.), Institute for Critical Technologies and Applied Science, Virginia Tech (P.R. and N.G.), and the Computational Tissue Engineering Interdisciplinary Graduate Education Program, Virginia Tech (T.M.M. and P.R.).
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N.G. performed statistical analysis, interpreted the data, and wrote the manuscript. L.W. performed the cellular work and assessments. K.A. performed the RNA-seq analysis and interpreted the results. Y.S. assisted with revisions and analyzing the RNA-seq data. P.N. assisted with revisions to the manuscript. T.M.M. and P.R. designed the scope of the paper, supervised, and wrote the paper.
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Gandhi, N., Wills, L., Akers, K. et al. Comparative transcriptomic and phenotypic analysis of induced pluripotent stem cell hepatocyte-like cells and primary human hepatocytes. Cell Tissue Res 396, 119–139 (2024). https://doi.org/10.1007/s00441-024-03868-9
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DOI: https://doi.org/10.1007/s00441-024-03868-9